Tetrahexin and process for preparing same

ABSTRACT

THE ANTIBIOTIC SUBSTANCE TETRAHEXIN, OF POLYENIC NATURE, OF HIGH ACTIVITY AGAINST PATHOGENIC MICROORGANISMS, AND THE METHOD OF PREPARATION OF THE SAME.

April 30, 1974 c v ETAL 3,808,331

TETRAHEXIN AND PROCESS FOR PREPARING SAME Filed Nov. 28, 1967 o o o o 9 oo o 1- N o o o I\ o O m WAVELENGTH (MICRONS) 1 1500 100,0 WAVENUMBER (CM O O O m 0 (o) 8 oo 8 3 8 5 0 (/0) EDNViiH/JSNVHJ. JANE/Inn:

XewA-ro (Reva-2r CAPom/n Coma/nu 79/570 Saw P 3,808,331 [Ce Patented Apr. 30, 1974 rings. The spores are cylindric in form and measure 2- 3,808,331 2.5a x 1-1-5/L. According to the classification of Pridham, TETRAHEXIN AND PROCESS FOR Heseltine and Benedict the strain belongs to the gray PREPARING SAME series and to the spire. section. Renato Craveri, Carolina Cornelli, and hero Sensr, Milan,

Italy, assignors to Lepetit, S.p.A., Milan, Italy Physiological and cultural characteristics Continuation-impart of abandoned application Ser. No.

266,182, Mar. 19, 1963. This application Nov. 28, 1967, The strain mesophilic and its growth requires: mini- Ser. No. 686,197 mum 12-14 C., optimum 2026 C. for the development Int. Cl- A61k 21/00 of the vegetative mycelium and 26-32 C. for the sporifica- 424-122 3 Clams 10 tion, maximum 34-36 C. It does not develop at pH 4.5

and 9, and has a growth optimum at about pH 7. ABSTRACT OF THE DISCLOSURE l Table presents the general physiologrcal and culture characterrstrcs of the strain, while Table II mdrcates the The a tl q q Substance tetra-begun, P p y nature utilization of various carbon sources using the base syn- Of hlgh actlvlty agamst Pathogemc mlcroorgamsms, and 15 thetic medium and the method of Pridham and Gottlieb. e of P p atw f the m The reported results were obtained after 15 days of incubatlOIl at 28 C.

TABLE Culture medium Vegetative mycelium 1 Aerial mycelium Soluble pigment Biochemical properties Agal Good growth, from intense Ash gray, moderate sporifi- From intense violet to dark violet to blackish brown. cation. brown well diffused. Bennet's agar Good growth, brow Absent Brown Hickey-Tresners a ar do Gray ijsh white, poor spend -do ca on. Oatmeal agar Good growth, from violet Ash gray, abundant sporifi- From lavender-gray to to brown. cation. brownish, well difiused. Czapek-glucose agar Good growth, light brown. A n Absent Nutrient agar Moderate growth colorle do do Nutrient glucose agar. Good growth, light brown do do Roux potato agar Good growth, colorless do do Roux carrot agar Moderate growth, coloress. do I r (in Skim-milk agar BB L .do do do Poor hydrolysis of casein. Peptone-iron agar Difco Poor growth, brow do Brownish Poor production of hydrogen sulfide. Synthetic-agar tyrosine Poor growth, colorless do Absent Not decomposed tyrosine. Agal-tyrosine Goedgrowth, violet brown. Grayish, poor sporification... Dark brown well (flu-88d..." Weak attack of tyrosine with some production of melar Y noid pigment. Synthetic agar cellulose Poor growth, colorless A n A n Not decomposed cellulose legal-cellulose G%od growth, blacksh Ash gray, poor sporification- Initially violet, then dark Do.

rown brown. Ca-malate agar Good growth, violet brown- Gray, poor sporification..... vrg gr wn, poorly Not decomposed Camalate.

' s a Nitrates broth, synthetic Very poor growth- A n A en Nirates not reduced to nitrites. Gelatine Poor surface grow h do Brown Not fluidified gelatlne. Agal-uric acid... Good growth 'violet Gray, poor sporlfication Intense violet well difiused Decomposed uric acid. Litmus-milk Diico Good surface grow h Absent Not evi n Positive peptonization,

negative coagulation,

1 Agal=Asparagine 1 g.; yeast extract Diico 1 g.; glycerol g.; soluble starch g.; agar g.; distilled water 1,000 ml; pH 7.2. This is a continuation-in-part of our copending applica- TABLE H tion Ser. No. 266,182 filed on Mar. 19, 1963, now aban domed. Carbon source Growth Sporlfication This invention relates to a new antibiotic substance and Arabinose Absent absent. to the process for preparing it. X

More particularly the invention is concerned with tetrahexin.

The process for producing tetraheXin comprises cultivating a strain of Streptomyces ATCC 14972 hereinafter described, in a culture medium containing assimilable sources of carbohydrates, nitrogen, and inorganic salts until substantial antibiotic activity is imparted to the culture medium, and recovering tetrahexin from the medium.

The strain Streptomyces ATCC 14972 has been isolated from a sample of soil collected in a chestnut wood in the surrounding of Genoa ad shows the characteristics hereinbelow described.

InositoL.

General charactensnc The new antibiotic is produced in high yield by sub- The colonies on solid medium appear powdery, often merged culture in fermentor and is recovered from the with wrinkled surface, of dark gray color, with diffused mycelium by extraction with a lower alkanol. The prodviolet brown pigment. The hyphae of the ripened aerial net is purified by counter-current distribution or solution mycelium become spirals of spore chains with many closed in acetic acid and precipitation by neutralization with 3 sodium hydroxidefrom the cooled solution-diluted 10: 1 with water.

Tetrahexin is soluble in dimethylformamide and acetic acid, scarcely soluble in lower alkanols (the solubility can be increased by adding to the alkanol 1-2% of calcium chloride) and in water at pH lower than3 and higher than9i;,insoluble in the other usual organic solvents, such as d'iethyl'ether, acetone, chloroform, benzene, and the like, and in water at pH 3-9. The antibiotic does not melt below 250 C. and decomposes above this temperature.

Tetrahexin is of polyenic nature and is'famphoteric; thefp K, of the two salificable functions as determined by spectrophotometry are 5.1 and 6.8. The antibiotic contains' nitrogen and is free from sulfur andhalogens; it

gives negative reaction to 'anthrone and ninhydrine, also after acid and alkaline hydrolysis, positive to Tollens and Fehling' reagets. It gives brown coloration with ferric. chloride and intense red-violet coloration with concenested sulfuric acid (reaction of polyenic substances). The elemental analysis carried out on samples purified from ac'eti c acid to constant extinction values at the UV spe" t'rum gives the following percent values: C, 65.55; H, 8. ,-N, 1.32; 0, 24.67 (by diiference).

The antibiotic tetrahexin shows in the UVacharacteristit; absorption spectrum with maximum values at and a'sho'uld'er at 335 mg. The maximum values of the UV spectrum indicate the presence of a tetraenic and 30 hexaenic chromophore. g

In the LR. region of the spectrum, which. is reported in FIG. 1, tetrahexin shows absorption maxima at the following wavelengths, indicated in cmf z 3300, 1700, 1650, 1625, 1600, 1295, 1255, 1043, 1003, 973, 723. The

spectrum was determined in a Perkin-Elmer apparatus on a sample suspended in mineral oil. 4 Samples of crude and purified product were subjected to paper chromatography using several solvent mixtures and the streaks were developed microbiologicallyon plates inoculated with Saccharomyces cerevisiae and Staphylococcus aureus, with all solvents systems mixtures only one inhibition one was noted and, the obtained Rfvalues were identical withboth microorganisms. The obtained results are summarized in Table III. l

Acetone-water 1:1. 84 0.81

Several countercurrent purification assayslwere made? ,two mixtures: (A) pyridine-ethylacetate-water (31516 5283 (B) butano -ligroin-phosphate buffer pH 9.Z-dimethyIfOrmamide-methanol (3 0.6 3 0.8 0.6). From the distribution curves it results that only one fraction was separated. with both mixtures and that the highest microbiologic activity is the one spectrophotometrically.

calculated both at 318 m (characteristic maximum-of thetetraenic chromophoric group) and at 350-.ma (characteristic maximum of the hexaenic chromophoric group).

The testtubes corresponding to the maximumxofthei, counter-current distribution performed with mixtureB;

were combined and a product was isolated which was subsequently submitted to 200 transfers with mixture A; the distribution curve shows again the presence of only one maximum. Both from chromatography and counter current distribution tetrahexin appears as a unitary antibiotic substance containing both the tetraenic and" hexaenic chro TAB LE IV MinimaL concentration of complete inhibition Microprganisms 11 Saccharomyces cerevisiae 0, O5 F' carlsber 0. 05 Sacchammyces ellipsoideas 0. O5 Saccharomyces ladwigii. 0. O5 K'Zoech era brevz's 0. 05 Torula utilz's. 0. 25' Rodotorala sp Q. 25 zygosaccharomyces glob ormts- '0. 1 u alts 0. 25 Cryptococcus neoforman 0. 25. C 0.25 Candida cruzez 0.-5 Altcman'a solam. 0. 1 Altem'arz'a citri 0. 1 Pnicillium'chrysogenum W Q, 176 '0; 25 Aspergillus niger 0. 25 Fusarium ozysporam var. lycopemcz. 0.5 Rz'zoctom'a solam'. 0. 25; Cercospara-acetosella 0. 25. Trichopht/to'n. mentagrophytes 0. 25 Trichosporon cutaneam 0. 1 Histoplasma capsulatum 0. 25 Nbcardia asteroides 0.25 Nocardia lutea 0. 1 Bacillus subtz'lisu 0. 05. Bacillus cereus var. mycoirles- O. 1 Staphylococcasaureua Oxf. ,0. 05 Sarci'aa lutea .0. 1 Oorynebacteria'm equin 0.1 Escherichia coli 200 Salmonella typhi-marz'um 200 Klcbs'tella 72" Proteus vulgarzs- L mophoric groups. Tetrahexin is stable for several months if preserved dry and in the dark. In aqueous solutions it is stable for some days only if stored in the dark at pH over 7. The bovine serum does not alter its microbiological activity. Tetrahexin is highly active against yeasts, moulds and gram-positive bacteria. It' possesses no activity -against gram-negative bacteria. i The antibiotic activity of tetrahexin as determinedby the agar streak method is shown in Table v I The antibiotic is dissolved in dimethylformamide. and the solutions to be used are consecutively prepared with distilled water. and polyvinylpyrrolidine :5. The used medium is pluriagar as described by Craveri et al., in Antibiotics and Chemotherapy 10, 430 (1960).

The given results were obtained after incubation at 28 C. for the test'microorganisms: 24 hoursfor bacteria and yeasts, 48 hours for filamentous fungi.

Thefaddition of bovine blood serum to "thelculture medium of pathogenic microorganisms does not impair serum at pH 7.0 at 22 C. after'7 hours.

Saccharomyce's cerevisiae -c 0.15 Caizdida albicaizs 0.1'5' Staphylococcus aarcus Oxf. I P Cryptbco'ccus nebformans 015 Tetrahexin possesses a low degree o ftoxicity my afii mals. The'acute toxicity data are hereinafter given' in' comparison with a widely used antibiotic substance polyenic nature; i.e. trichomycin.

LD on albino mice, in mg./k'g; A

Subcuta- Intraperineously toneally I Tetrahexin- 400 40 Tn'chomycin 4. 2-

Contrary to other, antibiotics of polyenic nature, tetra-' hexin is fairly stable at various pH values and tempera- 75 tures.

Solutions containing 200 /ml. of tetrahexin were prepared in a solvent consisting of 10 percent dimethylformamide, 20 percent methanol and 70 percent buffer at various pH. The -m.i.c. was then determined on four selected microorganisms at different pH values and temperatures. The obtained results are recorded in the following table.

M.i.c. in v/ml.

Saccha- Cryptoromycescoccus cerevi- Candida Staph. 'neofor- Sample siae albicans aureus mam Control solution at hour (zero) pH 7.0 25-0. 6 2. -1. 25 0. 6-0. 3 0.6-1. 25 1. Stability at various pH values:

pH 2.0 at 22 0., 7th hour- 5-2. 5 10 5-2. 5 10-5 pH 4.5 at 22 0., 7th hour- 0. 6-0. 3 2. 5-1. 25 0. 6-0. 3 0. 6-0. 3 pH 5.9 at 22 0., 7th hour- 0.3 0. 6 0. 3-0. 0. 3-0. 15 pH 7.0 at 22 0., 7th hour- 0.3 1. 25-0. 6 0.6-0.3 0. 6-0. 3 pH 8.0 at 22 0., 7th hour- 0. 6-0. 3 1. 25-0. 6 0. 6-0. 3 0. 6-0. 3 pH 9.2 at 22 0., 7th hour- 0. 6-0. 3 1. 25-0.6 0. 6 0.6 2. Stability at various temperatures.

pH 7.0 at37 C.,7th hour-- 0.6-0.3 1.250.6 0.3 0.3 pH 7.0 at 50 C.,7th h0ur-- 0.6-0.3 1. 25-0.6 0.6-0.3 0.6-0.3 pH 7.0 at 22 0., 7th hour- 1. 25-0.6 2. 5-1. 25 0.6 1. 25-0. 6 pH 7.0 at 22 0., 1 week- 1. 25-0. 6 2. 5-1. 25 1. 25-0. 6 0.6-0. 3 pH 7.0 at 2 C. 1 month..- -10 20-10 10-5 5 pH 7.0 at 22 0., 3 months.. 5-2. 5 5-2. 5 5-2. 5 5-2. 5 pH 7.0 at 4 0., 1 week.- 1. 25-0. 6 1. 25 2.5-1. 25 1. 25-0. 6 pH 7.0 at 4 C., 1 month 5 2.5 2. 5 2. 5-1 25 pH 7.0 at 4 C., 3 months...

1 Inactive at 20 'ylml.

The following nonlimitative example illustrate the invention.

EXAMPLE Five hundred millilitres flasks containing 100 ml. of medium A:

Yeast autolysate g 5 Meat extract a 5 Peptone -g-.. 5 Casein hydrolysate g 3 Glucose g 20 NaCl g 1.5 Distilled water ml 1000 pH after sterilization 6.8.

are inoculated with a spore suspension of Streptomyces ATCC 14972 after 48 hours of rotatory stirring at 28 C. and 250 r.p.m. the culture is used for inoculating 10 litre fermentors containing 4 litres of medium A.

After 24 hours fermentation (stirring 800 r.p.m., aeration 1 v./'v./m., temperature 24 C.) a fermentor is used as inoculum for five 20 litres production fermentors containing 10 litres of medium B:

pH after sterilization 6.6.

(stirring 1200 r.p.m., aeration 1.5 v./v./m. temperature 20 C.). After -85 hours fermentation the antibiotic concentration is assayed microbiologically on an ethanol extract of the mycelium (after filtration of the broth culture about of the produced active substance is retained in the mycelium) and shows 1446.000 dilution units on Saccharomyces cerevisiae ATCC 9763 and on Staphylococcus aureus 209 P. ATCC 6538 (assay method: agar streak).

The mycelium is separated by filtration at neutral pH with the addition of Celite, washed with water and dried under pressure. The obtained mass is extracted three times with ethanol and the combined ethanol extracts are concentrated in vacuo to /3 of the initial volume and allowed to stand 1 night at 40 C. The formed precipitate is filtered, thoroughly washed with acetone and dried in vacuo. From the mother liquors, by further concentration and cooling at 4 C. for 24 hours it is possible to obtain a second crop of antibiotic. From 10 litres of fermentation broth assaying 1416.000 dilution units 6.5 g. of active product, active at 0.5 'y/ml. on Saccharomyces cerevisiae are obtained.

We claim:

1. A process for preparing an antibiotic substance of polyenic nature, named tetrahexin, havingthe properties indicated in claim 3, which comprises cultivating under aerobic conditions the microorganism Streptomyces ATCC 14972 in an aqueous medium containing a source of carbon, a source of nitrogen and essential inorganic salts, until substantial antibiotic activity is imparted to the medium and recovering the antibiotic from the medium.

2. A process as in claim 1, wherein Streptomyces ATCC 14972 is cultivated in an aqueous medium containing 0.1 to 5.0% by weight of a nitrogenous substance, 0.5 to 5.0% by weight of a usable carbonaceous material, at a temperature between 20 C. and 35 C. and for about 24 to 48 hours while aerating the medium.

3. An antibiotic substance, obtainable by the process defined in claim 1, said substance being named tetrahexin and being soluble in dimethylformamide and in acetic acid, poorly soluble in lower alkanols and in water at a pH lower than 3 and higher than 9, insoluble in water at pH 3 to 9, said substance being polyenic and amphoteric in nature and having two salificable functions whose pKa are 5.1 and 6.8, its elemental analysis being C, 65.55%; H, 8.46%; N, 1.32%, and O, 24.67%, giving characteristic maxima at 368 mu the maximum values indicating the presence of a tetraenic and a hexaenic chromophore, said substance being active against yeast, molds, and gram-positive bacteria.

References Cited Derwent Farm Doc. #16924, French Pat. 1,395,876, published Apr. 16, 1965, pp. 477-484 (copy in Group JEROME D. GOLDBERG, Primary Examiner US. Cl. X.R. 

